Monoclonal antibodies directed to the HER2 receptor

ABSTRACT

A method of inhibiting growth of tumor cells which overexpress a growth factor receptor or growth factor by treatment of the cells with antibodies which inhibit the growth factor receptor function, is disclosed. A method of treating tumor cells with antibodies which inhibit growth factor receptor function, and with cytotoxic factor(s) such as tumor necrosis factor, is also disclosed. By inhibiting growth factor receptor functions tumor cells are rendered more susceptible to cytotoxic factors.

CROSS REFERENCES

This application is a continuation of U.S. application Ser. No.09/044,197 filed Mar. 17, 1998, now U.S. Pat. No. 6,165,464 which is acontinuation of U.S. application Ser. No. 08/447,478 filed May 23, 1995(now U.S. Pat. No. 5,772,997 issued Jun. 30, 1998), which is acontinuation of U.S. application Ser. No. 08/286,303 filed Aug. 5, 1994(now U.S. Pat. No. 5,677,171 issued Oct. 14, 1997), which is acontinuation of U.S. application Ser. No. 07/977,453 filed Nov. 18, 1992(now abandoned), which is a continuation of U.S. application Ser. No.07/147,461 filed Jan. 25. 1988 (now abandoned), which is acontinuation-in-part of U.S. application Ser. No. 07/143,912 filed Jan.12, 1988 (now abandoned), which applications are incorporated herein byreference and to which applications priority is claimed under 35 USC§120.

FIELD OF THE INVENTION

This invention is in the fields of immunology and cancer diagnosis andtherapy. More particularly it concerns antibodies specifically bindinggrowth factor receptors, hybridomas that produce these antibodies,immunochemicals made from the antibodies, and diagnostic methods thatuse the antibodies. The invention also relates to the use of theantibodies alone or in combination with cytotoxic factor(s) intherapeutic methods. Also encompassed by the invention is an assay fortyrosine kinases that are involved in tumorigenesis.

BACKGROUND OF THE INVENTION

Macrophages are one of the effector cell types that play an importantrole in immunosurveillance against neoplastic growth in vivo. In vitro,cell-mediated cytotoxicity requires selective binding between activatedmacrophages and target cells as well as the concomitant release ofcytotoxic factors. Some of the cytotoxic factors secreted by activatedmacrophages include reactive oxygen species such as the superoxide anionand hydrogen peroxide, arginase, interleukin 1, and tumor necrosisfactor-α (TNF-α). Acquired resistance to the toxic effects of thesefactors by tumor cells could be one mechanism which leads to the onsetand spread of tumor formation in vivo.

The observation that TNF-α can act as a potent effector of themacrophage-mediated antitumor response provides a rationale for its usein further studies on the regulation of tumorigenesis in vivo and tumorcell growth in vitro. The genes encoding TNF-α and TNF-β, a structurallyrelated cytotoxic protein formerly known as lymphotoxin, have beencloned and the corresponding proteins expressed in Escherichia coli.These proteins display an array of biological activities, includinginduction of hemorrhagic necrosis of Meth A sarcomas in vivo, inhibitionof the growth of certain tumor cells in vitro, synergistic enhancementof the in vitro anticellular effects of IFN-γ, activation of humanpolymorphonuclear neutrophil functions, and inhibition of lipidbiosynthesis. Recently, rHuTNF-α was shown to augment the growth ofnormal diploid fibroblasts in vitro. The divergent proliferativeresponses in the presence of rHuTNF-α are sometimes related tovariations in TNF binding.

Growth factors and their receptors are involved in the regulation ofcell proliferation and they also appear to play a key role inoncogenesis. Of the known proto-oncogenes, three are related to a growthfactor or a growth factor receptor. These genes include c-sis, which ishomologous to the transforming gene of the simian sarcoma virus and isthe B chain of platelet-derived growth factor (PDGF); c-fms, which ishomologous to the transforming gene of the feline sarcoma virus and isclosely related to the macrophage colony-stimulating factor receptor(CSF-1R); and c-erbB, which encodes the EGF receptor (EGFR) and ishomologous to the transforming gene of the avian erythroblastosis virus(v-erbB). The two receptor-related proto-oncogenes, c-fms and c-erbB,are members of the tyrosine-specific protein kinase family to which manyproto-oncogenes belong.

Recently, a novel transforming gene was identified as a result oftransfection studies with DNA from chemically induced ratneuroblastomas. This gene, called neu, was shown to be related to, butdistinct from, the c-erbB proto-oncogene. By means of v-erbB and humanEGFR as probes to screen human genomic and complementary DNA (cDNA)libraries, two other groups independently isolated human erbB-relatedgenes that they called HER2 and c-erbB-2 respectively. Subsequentsequence analysis and chromosomal mapping studies revealed thatc-erbB-2, and HER2 are species variants of neu. A fourth group, alsousing v-erbB as a probe, identified the same gene in a mammary carcinomacell line, MAC 117, where it was found to be amplified five- toten-fold.

This gene, which will be referred to herein as HER2, encodes a newmember of the tyrosine kinase family; and is closely related to, butdistinct from, the EGFR gene as reported by Coussens et al., Science230, 1132 (1985). HER2 differs from EGFR in that it is found on band q21of chromosome 17, as compared to band p11-p13 of chromosome 7, where theEGFR gene is located. Also, the HER2 gene generates a messenger RNA(MRNA) of 4.8 kb, which differs from the 5.8- and 10-kb transcripts forthe EGFR gene. Finally, the protein encoded by the HER2 gene is 185,000daltons, as compared to the 170,000-dalton protein encoded by the EGFRgene. Conversely, on the basis of sequence data, HER2 is more closelyrelated to the EGFR gene than to other members of the tyrosine kinasefamily. Like the EGFR protein, the HER2 protein (p185) has anextracellular domain, a transmembrane domain that includes twocysteine-rich repeat clusters, and an intracellular kinase domain,indicating that it is likely to be a cellular receptor for an as yetunidentified ligand. HER2 p185 is referred to as p185 or the HER2receptor herein.

Southern analysis of primary human tumors and established tumor-derivedcell lines revealed amplification and in some cases rearrangement of theEGF receptor gene. Amplification was particularly apparent in squamouscarcinomas and glioblastomas. The HER2 gene was also found to beamplified in a human salivary gland adenocarcinoma, a renaladenocarcinoma, a mammary gland carcinoma, and a gastric cancer cellline. Recently, Slamon et al., Science 235, 177 (1987) demonstrated thatabout 30% of primary human breast carcinoma tumors contained anamplified HER2 gene. Although a few sequence rearrangements weredetected, in most tumors there were no obvious differences betweenamplified and normal HER2 genes. Furthermore, amplification of the HER2gene correlated significantly with the negative prognosis of the diseaseand the probability of relapse.

To investigate the significance of the correlation betweenoverexpression and cellular transformation as it has been observed forproto-oncogenes c-mos and N-myc, a HER2 expression vector and aselection scheme that permitted sequence amplification aftertransfection of mouse NIH 3T3 cells was employed by Hudziak et al.,Proc, Natl. Acad. Sci. (USA) 84, 7159 (1987). Amplification of theunaltered HER2 gene in NIH 3T3 cells lead to overexpression of p185 aswell as cellular transformation and tumor formation in athymic mice.

The effects of antibodies specifically binding growth factors or growthfactor receptors has been studied. Examples are discussed below.

Rosenthal et al., Cell 46, 301 (1986) introduced a human TGF-α cDNAexpression vector into established non-transformed rat fibroblast cells.Synthesis and secretion of TGF-α by these cells resulted in loss ofanchorage-dependent growth and induced tumor formation in nude mice.Anti-human TGF-α monoclonal antibodies prevented the rat cells fromforming colonies in soft agar, i.e. loss of anchorage dependence. Gillet al. in J. Biol. Chem. 259, 7755 (1984) disclose monoclonal antibodiesspecific for EGF receptor which were inhibitors of EGF binding andantagonists of EGF-stimulated tyrosine protein kinase activity.

Drebin et al. in Cell 41, 695 (1985) demonstrated that exposure of aneu-oncogene-transformed NIH 3T3 cell to monoclonal antibodies reactivewith the neu gene product, cause the neu-transformed NIH 3T3 cell torevert to a non-transformed phenotype as determined by anchorageindependent growth. Drebin et al. in Proc. Natl. Acad. Sci. 83, 9129(1986) demonstrated that in vivo treatment with a monoclonal antibody(IgG2a isotype) specifically binding the protein encoded by the neuoncogene significantly inhibited the tumorigenic growth ofneu-transformed NIH 3T3 cells implanted into nude mice.

Akiyama et al. in Science 232, 1644 (1986) raised antibodies against asynthetic peptide corresponding to 14 amino acid residues at thecarboxy-terminus of the protein deduced from the c-erbB-2 (HER2)nucleotide sequence.

Growth factors have been reported to interact in both a synergistic andan antagonistic manner. For example, TGF-α and TGF-β synergisticallyenhance the growth of NRK-49F fibroblasts, whereas PDGF down regulatesEGF receptor function on 3T3 cells. A variety of transformed cellssecrete factors which are believed to stimulate growth by an autocrinemechanism. Sugarman et al., Cancer Res. 47, 780 (1987) demonstrated thatunder certain conditions, growth factors can block the antiproliferativeeffects of TNF-α on sensitive tumor cells. Specifically, epidermalgrowth factor (EGF) and recombinant human transforming growth factor-α(rHuTGF-α) were shown to interfere with the in vitro antiproliferativeeffects of recombinant human tumor necrosis factor-α (rHuTNF-α) and -βon a human cervical carcinoma cell line, ME-180. The inhibitory effectcould be observed at EGF or rHuTGF-α concentrations of 0.1 to 100 ng/ml,and was maximal between 1 and 10 ng/ml. This response was apparently notdue to down regulation of the TNF receptor or to alteration of theaffinity of TNF-α for its receptor. Since the antiproliferative effectof recombinant human interferon-γ was not significantly affected by thepresence of EGF or rHuTGF-α, the inhibition was specific for recombinantTNFs and was not due solely to enhanced proliferation induced by thegrowth factors. Neither growth factor had a substantial protectiveeffect on the synergistic cytotoxicity observed when tumor cells wereexposed simultaneously to rHuTNF-α and recombinant human interferon-γ.TGF-β can also interfere with the antiproliferative effects of rHuTNF-αin vitro. At concentrations of less than 1 ng/ml, TGF-β significantlyantagonized the cytotoxic effects of rHuTNF-α on NIH 3T3 fibroblasts.Since EGF, platelet-derived growth factor, and TGF-β all enhanced NIH3T3 cell proliferation, but only TGF-β interfered with rHuTNF-αcytotoxicity, the protective effects of TGF-β were not related in asimple manner to enhanced cell proliferation. rHuTGF-α and TGF-β did nothave a significant protective effect against rHuTNF-α-mediatedcytotoxicity on two other tumor cell lines, BT-20 and L-929 cells.

It is an object of the subject invention to provide antibodies capableof inhibiting growth factor receptor function.

It is a further object of the invention to provide an improved assay forthe HER2 receptor.

It is a further object of the invention to provide improved methods oftumor therapy.

It is a further object of the invention to provide a method ofinhibiting the growth of tumor cells which overexpress a growth factorreceptor and/or growth factor.

It is a further object of the invention to provide a method for treatinga tumor by treatment of the tumor cells with antibodies capable ofinhibiting growth factor receptor function, and with cytotoxic factorssuch as tumor necrosis factor.

A still further object of the invention is to provide an assay fortyrosine kinases that may have a role in tumorigenesis.

Other objects, features and characterisitics of the present inventionwill become apparent upon consideration of the following description andthe appended claims.

SUMMARY OF THE INVENTION

The subject invention relates to monoclonal antibodies specificallybinding the external domain of the HER2 receptor. The invention alsorelates to an assay for the HER2 receptor comprising exposing cells toantibodies specifically binding the extracellular domain of the HER2receptor, and determining the extent of binding of said antibodies tosaid cells. Another embodiment of the invention relates to a method ofinhibiting growth of tumor cells by administering to a patient atherapeutically effective amount of antibodies capable of inhibiting theHER2 receptor function. A further embodiment of the invention relates toadministering a therapeutically effective amount of antibodies capableof inhibiting growth factor receptor function, and a therapeuticallyeffective amount of a cytotoxic factor. A still further embodiment ofthe invention is an assay for tyrosine kinases that may have a role intumorigenesis comprising exposing cells suspected to be TNF-α resistantto TNF-α, isolating those cell which are TNF-α resistant, screening theisolated cells for increased tyrosine kinase activity, and isolatingreceptors and other proteins having increased tyrosine kinase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows TNF-α resistance of NIH 3T3 cells expressing variouslevels of HER2 p185. FIG. 1b shows macrophage cytotoxicity assays forNIH 3T3 cells expressing various levels of HER2 p185.

FIG. 2 demonstrates the level of TNF-α binding for a control cell line(NIH 3T3 neo/dhfr) and for a cell line overexpressing HER2 p185(HER2-3₈₀₀).

FIG. 3 shows inhibition of SK BR3 cell growth by anti-HER2 monoclonalantibodies.

FIG. 4 is a dose response curve comparing the effect of an irrelevantmonoclonal antibody (anti-HBV) and the effect of monoclonal antibody 4D5(anti-HER2) on the growth of SK BR3 cells in serum.

FIGS. 5a, 5 b and 6 a show percent viability of SK BR3 cells as afunction of increasing TNF-α concentration and anti-HER2 p185 monoclonalantibody concentration. Each Figure shows the results for a differentanti-HER2 p185 monoclonal antibody. FIG. 6b is a control using anirrelevant monoclonal antibody. In FIGS. 5a, 5 b, 6 a and 6 b, —represents TNF-α alone; ▪—▪ represents antibody (Ab) alone; ∘—∘represents 100 U/ml TNF-α and ΔAb; □—□ represents 1000 U/ml TNF-α andΔAb; and Δ—Δ represents 10,000 U/ml TNF-α and ΔAb.

FIG. 7 shoes percent viability of MDA-MB-175-VII cells as a function ofincreasing TNF-α concentration and anti-HER2 p185 monoclonal antibodyconcentration. In FIG. 7, — represents TNF-α alone; ▪—▪ representsantibody (Ab) alone; ∘—∘ represents 100 U/ml TNF-α and ΔAb; □—□represents 1000 U/ml TNF-α and ΔAb; and Δ—Δ represents 10,000 U/ml TNF-αand ΔAb.

FIG. 8 shows percent viability of NIH 3T3 cells overexpressing HER2 p185as a function of increasing TNF-α concentration and anti-HER2 p185monoclonal antibody concentration. In FIG. 8, — represents TNF-αalone; ▪—▪ represents antibody (Ab) alone; ∘—∘ represents 100 U/ml TNF-αand ΔAb; □—□ represents 1000 U/ml TNF-α and ΔAb; and Δ—Δ represents10,000 U/ml TNF-α and ΔAb.

DETAILED DESCRIPTION OF THE INVENTION

A new application of antibodies to inhibit the growth of tumor cells hasbeen discovered. Surprisingly it has been found that by inhibitinggrowth factor receptor function, e.g. the HER2 receptor function, cellgrowth is inhibited, and the cells are rendered more susceptible tocytotoxic factors. Thus, for example, breast cancer cells which arerefractory to TNF-α alone can be made susceptible to TNF-α if the cellsare first treated with antibodies which inhibit growth factor receptorfunction. The increase of susceptibility has been demonstrated using theHER2 receptor and monoclonal antibodies directed against the HER2receptor, and tumor necrosis factor-α.

The method of this invention is useful in the therapy of malignant orbenign tumors of mammals where the abnormal growth rate of the tumor isdependent upon growth factor receptors. Abnormal growth rate is a rateof growth which is in excess of that required for normal homeostasis andis in excess of that for normal tissues of the same origin. Many ofthese tumors are dependent upon extracellular sources of the growthfactor recognized by the receptor, or upon synthesis of the growthfactor by the tumor cell itself. This latter phenomenon is termed“autocrine” growth.

The methods of the subject invention is applicable where the followingconditions are met:

(1) the growth factor receptor and/or ligand (growth factor) isexpressed, and tumor cell growth depends upon the growth factor receptorbiological function;

(2) antibodies specifically binding the growth factor receptor and/orligand inhibit the growth factor receptor biological function.

While not wishing to be constrained to any particular theory ofoperation of the invention, it is believed that the antibodies inhibitgrowth factor receptor biological function in one or more of thefollowing ways:

(a) The antibodies bind to the extracellular domain of the receptor andinhibit the ligand from binding the receptor;

(b) The antibodies bind the ligand (the growth factor) itself andinhibit the ligand from binding the receptor;

(c) The antibodies down regulate the growth factor receptor;

(d) The antibodies sensitize tumor cells to the cytotoxic effects of acytotoxic factor such as TNF-α;

(e) The antibodies inhibit the tyrosine kinase activity of the receptor.

In cases (f) and (g), the antibodies inhibit growth factor receptorbiological function indirectly by mediating cytotoxicity via a targetingfunction:

(f) The antibodies belong to a sub-class or isotype that upon complexingwith the receptor activates serum complement and/or mediateantibody-dependent cellular cytotoxicity (ADCC), e.g. IgG2a antibodies;

(g) The antibodies which bind the receptor or growth factor areconjugated to a toxin (immunotoxins);

Advantageously antibodies are selected which greatly inhibit thereceptor function by binding the steric vicinity of the ligand bindingsite of the receptor (blocking the receptor), and/or which bind thegrowth factor in such a way as to prevent (block) the ligand frombinding to the receptor. These antibodies are selected usingconventional in vitro assays for selecting antibodies which neutralizereceptor function. Antibodies that act as ligand agonists by mimickingthe ligand are discarded by conducting suitable assays as will beapparent to those skilled in the art. For certain tumor cells, theantibodies inhibit an autocrine growth cycle (i.e. where a cell secretesa growth factor which then binds to a receptor of the same cell). Sincesome ligands, e.g. TGF-α, are found lodged in cell membranes, theantibodies serving a targeting function are directed against the ligandand/or the receptor.

Certain tumor cells secrete growth factors that are required for normalcellular growth and division. These growth factors, however, can undersome conditions stimulate unregulated growth of the tumor cell itself,as well as adjacent non-tumor cells, and can cause a tumor to form.

Epidermal Growth Factor (EGF) has dramatic stimulatory effects on cellgrowth. In purified receptor preparations, the EGF receptor is a proteinkinase that is activated by the binding of EGF. Substrate proteins forthis kinase are phosphorylated on tyrosine residues. The receptors forinsulin, platelet-derived growth factor (PDGF) and other growth hormonesalso are tyrosine-specific kinases. It is believed that ligand bindingto the receptor triggers phosphorylation of certain proteins by thereceptor and in this way stimulates cell growth. About one-third of theknown oncogenes encode proteins that phosphorylate tyrosine residues onother proteins. It is believed that these oncogene products triggerresponses analogous to the responses of cells to growth factors andhormones. The erbB oncogene product is a portion of the EGF receptorthat lacks the hormone-binding domain and may give rise to aconstitutive growth-stimulating signal.

One embodiment of this invention is a method of inhibiting the growth oftumor cells by administering to a patient a therapeutically effectiveamount of antibodies that inhibit the HER2 receptor biological functionof tumor cells.

Overexpression of growth factor receptors increases the resistance ofcells to TNF as demonstrated below. Overexpression of the HER1 receptor(EGF receptor), met receptor-like protooncogene product, and HER2receptor all show this increased resistance. It is shown in the Examplesbelow that amplified expression of HER2, which encodes the HER2 receptor(p185), induces resistance of NIH 3T3 cells to the cytotoxic effects ofmacrophages or TNF-α. Induction of NIH 3T3 cell resistance to TNF-α byoverexpression of p185 is accompanied by alterations in the binding ofTNF-α to its receptor. Overexpression of p185 is also associated withresistance of certain human breast tumor cell lines to the cytotoxiceffects of TNF-α.

In another embodiment of the invention, tumor cells are treated by (1)administering to a patient antibodies directed against the growth factorand/or its receptor, that inhibit the biological function of thereceptor and that sensitize the cells to cytotoxic factors such as TNF,and (2) administering to the patient cytotoxic factor(s) or otherbiological response modifiers which activate immune system cellsdirectly or indirectly to produce cytotoxic factors.

The cytotoxic factor, such as TNF-α, exerts its cytostatic (cell growthsuppressive) and cytotoxic (cell destructive) effect. Examples of usefulcytotoxic factors are TNF-α, TNF-β, IL-1, INF-γ and IL-2, andchemotherapeutic drugs such as 5FU, vinblastine, actinomycin D,etoposide, cisplatin, methotrexate, and doxorubicin. Cytotoxic factorscan be administered alone or in combination. In a still furtherembodiment of the invention, the patient is treated with antibodieswhich inhibit receptor function, and with autologous transfer therapy,e.g. LAK or TIL cells.

Tumor necrosis factors are polypeptides produced by mitogen-stimulatedmacrophages or lymphocytes which are cytotoxic for certain malignantlytransformed cells. The anti-tumor effect of TNF-α is known to besynergistically potentiated by interferons. The anti-tumor effect ofTNF-α and TNF-β in admixture are additive, as are the antiviral effectsof interferons alpha and beta.

The tumor necrosis factors include TNF-α and TNF-β. The former isdescribed together with methods for its synthesis in recombinant cellculture, in U.S. Pat. No. 4,650,674, in copending U.S. Ser. No. 881,311,filed Jul. 2, 1986, and in European Patent Application 0168214; thelatter is described in European Patent Application 0164965. Each of thedocuments is hereby incorporated by reference. The TNF-α and TNF-βdescribed in these patent documents includes cytotoxic amino acidsequence and glycosylation variants. TNF-α and TNF-β fromnon-recombinant sources are also useful in the method of this invention.

The preferred TNF is mature human TNF-α from recombinant microbial cellculture. The TNF ordinarily will have a cytolytic activity onsusceptible L-M murine cells of greater than about 1×10⁶ units/mg,wherein a unit is defined as set forth in the above-described patentapplication.

In another embodiment of the subject invention, one or more additionalcytokines and/or cytotoxic factors are administered with TNF-α, egs.interferons, interleukins, and chemotherapeutic drugs.

The compositions herein include a pharmaceutically acceptable vehicle,such as those heretofore used in the therapeutic administration ofinterferons or TNF, e.g. physiological saline or 5% dextrose, togetherwith conventional stabilizers and/or excipients such as human serumalbumin or mannitol. The compositions are provided lyophilized or in theform of sterile aqueous solutions.

Several variables will be taken into account by the ordinary artisan indetermining the concentration of TNF in the therapeutic compositions andthe dosages to be administered. Therapeutic variables also include theadministration route, and the clinical condition of the patient.

The cytotoxic factor(s) and antibodies inhibiting growth factor receptorfunction are administered together or separately. If the latter,advantageously the antibodies are administered first and the TNFthereafter within 24 hours. It is within the scope of this invention toadminister the TNF and antibodies in multiple cycles, depending upon theclinical response of the patient. The TNF and antibodies areadministered by the same or separate routes, for example by intravenous,intranasal or intramuscular administration.

The method of the subject invention can be used with tumor cells whichoverexpress growth factor receptor and/or ligand where antibodies can beproduced which inhibit the growth factor receptor function. A cell (e.g.breast tumor cell) overexpresses a growth factor receptor if the numberof receptors on the cell exceeds the number on the normal healthy cell(e.g. normal breast tissue cell). Examples of carcinomas where the HER2receptor is overexpressed (and thus the method of the subject inventionis applicable), are human breast, renal, gastric and salivary glandcarcinomas.

A further embodiment of the invention is an assay for identifyingreceptors and other proteins having increased tyrosine kinase activity,and for identifying oncogenes that transform cells. Amplification ofcertain oncogenes encoding tyrosine kinases correlates with TNF-αresistance. If cells are selected for resistance to TNF-α, some of thesewill have increased tyrosine kinase activity. Some of the tyrosinekinases will be receptors. The genes encoding the tyrosine kinases arethen cloned using standard techniques for the cloning of genes.Identification of the receptor or other protein permits the design ofreagents which inhibit receptor (or other protein) function and inducecellular sensitivity to cytotoxic factors as demonstrated herein withHER2. Identification of the receptor also permits subsequentidentification of the receptor's ligand. The assay comprises exposingcells suspected to be TNF-α sensitive to TNF-α, and isolating thosecells which are TNF-α resistant. The TNF-α resistant cells are thenscreened for increased tyrosine kinase activity, and receptors and otherproteins having increased tyrosine kinase activity are isolated.

Antibodies

In accordance with this invention, monoclonal antibodies specificallybinding growth factors or growth factor receptors such as the HER2receptor, were isolated from continuous hybrid cell lines formed by thefusion of antigen-primed immune lymphocytes with myeloma cells.Advantageously, the monoclonal antibodies of the subject invention whichbind growth factor receptors, bind the extracellular domain of thereceptors. In another embodiment of the invention, polyclonal antibodiesspecifically binding the growth factors or growth factor receptors areused.

The antibodies of the subject invention which are used in tumor therapyadvantageously inhibit tumor cell growth greater than 20%, and mostadvantageously greater than 50%, in vitro. These antibodies are obtainedthrough screening (see, for example, the discussion relating to FIG. 3).The anti-HER2 receptor monoclonal antibodies of the subject inventionwhich are used in tumor therapy are capable of inhibiting serumactivation of the receptor.

Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to conventional antibody(polyclonal) preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.Monoclonal antibodies are useful to improve the selectivity andspecificity of diagnostic and analytical assay methods usingantigen-antibody binding. A second advantage of monoclonal antibodies isthat they are synthesized by the hybridoma culture, uncontaminated byother immunoglobulins. Monoclonal antibodies may be prepared fromsupernatants of cultured hybridoma cells or from ascites induced byintra-peritoneal inoculation of hybridoma cells into mice.

The hybridoma technique described originally by Kohler and Milstein,Eur. J. Immunol. 6, 511 (1976) has been widely applied to produce hybridcell lines that secrete high levels of monoclonal antibodies againstmany specific antigens.

The route and schedule of immunization of the host animal or culturedantibody-producing cells therefrom are generally in keeping withestablished and conventional techniques for antibody stimulation andproduction. Applicants have employed mice as the test model although itis contemplated that any mammalian subject including human subjects orantibody producing cells therefrom can be manipulated according to theprocesses of this invention to serve as the basis for production ofmammalian, including human, hybrid cell lines.

After immunization, immune lymphoid cells are fused with myeloma cellsto generate a hybrid cell line which can be cultivated and subcultivatedindefinitely, to produce large quantities of monoclonal antibodies. Forpurposes of this invention, the immune lymphoid cells selected forfusion are lymphocytes and their normal differentiated progeny, takeneither from lymph node tissue or spleen tissue from immunized animals.Applicants prefer to employ immune spleen cells, since they offer a moreconcentrated and convenient source of antibody producing cells withrespect to the mouse system. The myeloma cells provide the basis forcontinuous propagation of the fused hybrid. Myeloma cells are tumorcells derived from plasma cells.

It is possible to fuse cells of one species with another. However, it ispreferred that the source of immunized antibody producing cells andmyeloma be from the same species.

The hybrid cell lines can be maintained in culture in vitro in cellculture media. The cell lines of this invention can be selected and/ormaintained in a composition comprising the continuous cell line in theknown hypoxanthine-aminopterin-thymidine (HAT) medium. In fact, once thehybridoma cell line is established, it can be maintained on a variety ofnutritionally adequate media. Moreover, the hybrid cell lines can bestored and preserved in any number of conventional ways, includingfreezing and storage under liquid nitrogen. Frozen cell lines can berevived and cultured indefinitely with resumed synthesis and secretionof monoclonal antibody. The secreted antibody is recovered from tissueculture supernatant by conventional methods such as precipitation, ionexchange chromatography, affinity chromatography, or the like.

While the invention is demonstrated using mouse monoclonal antibodies,the invention is not so limited; in fact, human antibodies may be usedand may prove to be preferable. Such antibodies can be obtained by usinghuman hybridomas (Cole et al., Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, p. 77 (1985)). In fact, according to the invention,techniques developed for the production of “chimeric antibodies”(Morrison et al., Proc. Natl. Acad. Sci. 81, 6851 (1984); Neuberger etal., Nature 312, 604 (1984); Takeda et al., Nature 314, 452 (1985)) bysplicing the genes from a mouse antibody molecule of appropriate antigenspecificity together with genes from a human antibody molecule ofappropriate biological activity (such as ability to activate humancomplement and mediate ADCC) can be used; such antibodies are within thescope of this invention.

As another alternative to the cell fusion technique, EBV immortalized Bcells are used to produce the monoclonal antibodies of the subjectinvention. Other methods for producing monoclonal antibodies such asrecombinant DNA, are also contemplated.

The immunochemical derivatives of the antibodies of this invention thatare of prime importance are (1) immunotoxins (conjugates of the antibodyand a cytotoxic moiety) and (2) labeled (e.g. radiolabeled,enzyme-labeled, or fluorochrome-labeled) derivatives in which the labelprovides a means for identifying immune complexes that include thelabeled antibody. The antibodies are also used to induce lysis throughthe natural complement process, and to interact with antibody dependentcytotoxic cells normally present.

Immunotoxins

The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or anenzymatically active toxin of bacterial or plant origin, or anenzymatically active fragment (“A chain”) of such a toxin. Enzymaticallyactive toxins and fragments thereof used are diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaccaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, and enomycin. In anotherembodiment, the antibodies are conjugated to small molecule anticancerdrugs. Conjugates of the monoclonal antibody and such cytotoxic moietiesare made using a variety of bifunctional protein coupling agents.Examples of such reagents are SPDP, IT, bifunctional derivatives ofimidoesters such a dimethyl adipimidate HCl, active esters such asdisuccinimidyl suberate, aldehydes such as glutaraldehyde, bis-azidocompounds such as bis (p-azidobenzoyl) hexanediamine, bis-diazoniumderivatives such as bis-(p-diazoniumbenzoyl)-ethylenediamine,diisocyanates such as tolylene 2,6-diisocyanate, and bis-active fluorinecompounds such as 1,5-difluoro-2,4-dinitrobenzene. The lysing portion ofa toxin may be joined to the Fab fragment of the antibodies.

Advantageously, monoclonal antibodies specifically binding the externaldomain of the target growth factor receptor, e.g. HER2 receptor, areconjugated to ricin A chain. Most advantageously the ricin A chain isdeglycosylated and produced through recombinant means. An advantageousmethod of making the ricin immunotoxin is described in Vitetta et al.,Science 238, 1098 (1987) hereby incorporated by reference.

When used to kill human cancer cells in vitro for diagnostic purposes,the conjugates will typically be added to the cell culture medium at aconcentration of at least about 10 nM. The formulation and mode ofadministration for in vitro use are not critical. Aqueous formulationsthat are compatible with the culture or perfusion medium will normallybe used. Cytotoxicity may be read by conventional techniques todetermine the presence or degree of cancer.

Cytotoxic radiopharmaceuticals for treating cancer may be made byconjugating radioactive isotopes (e.g. I, Y, Pr) to the antibodies. Theterm “cytotoxic moiety” as used herein is intended to include suchisotopes.

In another embodiment, liposomes are filled with a cytotoxic drug andthe liposomes are coated with antibodies specifically binding a growthfactor receptor. Since there are many receptor sites, this methodpermits delivery of large amounts of drug to the correct cell type.

Antibody Dependent Cellular Cytotoxicity

The present invention also involves a method based on the use ofantibodies which are (a) directed against growth factor receptors suchas HER2 p185, and (b) belong to a subclass or isotype that is capable ofmediating the lysis of tumor cells to which the antibody molecule binds.More specifically, these antibodies should belong to a subclass orisotype that, upon complexing with growth factor receptors, activatesserum complement and/or mediates antibody dependent cellularcytotoxicity (ADCC) by activating effector cells such as natural killercells or macrophages.

The present invention is also directed to the use of these antibodies,in their native form, for therapy of human tumors. For example, manyIgG2a and IgG3 mouse antibodies which bind tumor-associated cell surfaceantigens can be used in vivo for tumor therapy. In fact, since HER2 p185is present on a variety of tumors, the subject antibodies and theirtherapeutic use have general applicability.

Biological activity of antibodies is known to be determined, to a largeextent, by the Fc region of the antibody molecule (Uananue andBenacerraf, Textbook of Immunology, 2nd Edition, Williams & Wilkins, p.218 (1984)). This includes their ability to activate complement and tomediate antibody-dependent cellular cytotoxicity (ADCC) as effected byleukocytes. Antibodies of different classes and subclasses differ inthis respect, and, according to the present invention, antibodies ofthose classes having the desired biological activity are selected. Forexample, mouse immunoglobulins of the IgG3 and IgG2a class are capableof activating serum complement upon binding to the target cells whichexpress the cognate antigen.

In general, antibodies of the IgG2a and IgG3 subclass and occasionallyIgG1 can mediate ADCC, and antibodies of the IgG3, and IgG2a and IgMsubclasses bind and activate serum complement. Complement activationgenerally requires the binding of at least two IgG molecules in closeproximity on the target cell. However, the binding of only one IgMmolecule activates serum complement.

The ability of any particular antibody to mediate lysis of the tumorcell target by complement activation and/or ADCC can be assayed. Thetumor cells of interest are grown and labeled in vivo; the antibody isadded to the tumor cell culture in combination with either serumcomplement or immune cells which may be activated by the antigenantibody complexes. Cytolysis of the target tumor cells is detected bythe release of label from the lysed cells. In fact, antibodies can bescreened using the patient's own serum as a source of complement and/orimmune cells. The antibody that is capable of activating complement ormediating ADCC in the in vitro test can then be used therapeutically inthat particular patient.

Antibodies of virtually any origin can be used according to thisembodiment of the present invention provided they bind growth factorreceptors such as HER2 p185 and can activate complement or mediate ADCC.Monoclonal antibodies offer the advantage of a continuous, ample supply.In fact, by immunizing mice with, for example, HER2 p185, establishinghybridomas making antibodies to p185 and selecting hybridomas makingantibodies which can lyse tumor cells in the presence of humancomplement, it is possible to rapidly establish a panel of antibodiescapable of reacting with and lysing a large variety of human tumors.

Therapeutic Uses of the Antibodies

When used in vivo for therapy, the antibodies of the subject inventionare administered to the patient in therapeutically effective amounts(i.e. amounts that eliminate or reduce the patient's tumor burden). Theywill normally be administered parenterally, when possible, at the targetcell site, or intravenously. The dose and dosage regimen will dependupon the nature of the cancer (primary or metastatic), its population,the site to which the antibodies are to be directed, the characteristicsof the particular immunotoxin (when used), e.g., its therapeutic index,the patient, and the patient's history. The amount of antibodyadministered will typically be in the range of about 0.1 to about 10mg/kg of patient weight.

For parenteral administration the antibodies will be formulated in aunit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutically acceptable. parenteral vehicle. Suchvehicles are inherently nontoxic, and non-therapeutic. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils and ethyloleate may also be used. Liposomes may be used as carriers. The vehiclemay contain minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives. Theantibodies will typically be formulated in such vehicles atconcentrations of about 1 mg/ml to 10 mg/ml.

The selection of an antibody subclass for therapy will depend upon thenature of the tumor antigen. For example, an IgM may be preferred insituations where the antigen is highly specific for the tumor target andrarely occurs on normal cells. However, where the tumor-associatedantigen is also expressed in normal tissues, albeit at much lowerlevels, the IgG subclass may be preferred for the following reason:since the binding of at least two IgG molecules in close proximity isrequired to activate complement, less complement mediated damage mayoccur in the normal tissues which express smaller amounts of the antigenand, therefore, bind fewer IgG antibody molecules. Furthermore, IgGmolecules by being smaller may be more able than IgM molecules tolocalize to tumor tissue.

There is evidence that complement activation in vivo leads to a varietyof biological effects, including the induction of an inflammatoryresponse and the activation of macrophages (Uananue and Benecerraf,Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)).Tumor cells are more sensitive to a cytolytic effect of activatedmacrophages than are normal cells, Fidler and Poste, Springer Semin.Immunopathol. 5, 161 (1982). The increased vasodilation accompanyinginflammation may increase the ability of various anti-cancer agents,such as chemotherapeutic drugs, radiolabelled antibodies, etc., tolocalize in tumors. Therefore, antigen-antibody combinations of the typespecified by this invention can be used therapeutically in many ways andmay circumvent many of the problems normally caused by the heterogeneityof tumor cell populations. Additionally, purified antigens (Hakomori,Ann. Rev. Immunol. 2, 103 (1984)) or anti-idiotypic antibodies (Nepom etal., Proc. Natl. Acad. Sci. 81, 2864 (1985); Koprowski et al., Proc.Natl. Acad. Sci. 81, 216 (1984)) relating to such antigens could be usedto induce an active immune response in human cancer patients. Such aresponse includes the formation of antibodies capable of activatinghuman complement and mediating ADCC and by such mechanisms cause tumordestruction.

Immunoassays

Described herein are serological methods for determining the presence ofHER2 p185. Essentially, the processes of this invention compriseincubating or otherwise exposing the sample to be tested to monoclonalantibodies and detecting the presence of a reaction product. Thoseskilled in the art will recognize that there are many variations ofthese basic procedures. These include, for example, RIA, ELISA,precipitation, agglutination, complement fixation andimmuno-fluorescence. In the currently preferred procedures, themonoclonal antibodies are appropriately labeled.

The labels that are used in making labeled versions of the antibodiesinclude moieties that may be detected directly, such as radiolabels andfluorochromes, as well as moieties, such as enzymes, that must bereacted or derivatized to be detected. The radiolabel can be detected byany of the currently available counting procedures. The preferredisotope labels are ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, 32P and ³⁵S. The enzymelabel can be detected by any of the currently utilized colorimetric,spectrophotometric, fluorospectro-photometric or gasometric techniques.The enzyme is combined with the antibody with bridging molecules such ascarbodiimides, periodate, diisocyanates, glutaraldehyde and the like.Many enzymes which can be used in these procedures are known and can beutilized. Examples are peroxidase, alkaline phosphatase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plus peroxidase, galactose oxidase plus peroxidase and acidphosphatase. Fluorescent materials which may be used include, forexample, fluorescein and its derivatives, rhodamine and its derivatives,auramine, dansyl, umbelliferone, luciferia,2,3-dihydrophthalazinediones, horseradish peroxidase, alkalinephosphatase, lysozyme, and glucose-6-phosphate dehydrogenase. Theantibodies may be tagged with such labels by known methods. Forinstance, coupling agents such as aldehydes, carbodiimides, dimaleimide,imidates, succinimides, bid-diazotized benzadine and the like may beused to tag the antibodies with the above-described fluorescent,chemiluminescent, and enzyme labels. Various labeling techniques aredescribed in Morrison, Methods in Enzymology 32b, 103 (1974), Syvanen etal., J. Biol. Chem. 284, 3762 (1973) and Bolton and Hunter, Biochem J.133, 529(1973) hereby incorporated by reference.

The antibodies and labeled antibodies may be used in a variety ofimmunoimaging or immunoassay procedures to detect the presence of cancerin a patient or monitor the status of such cancer in a patient alreadydiagnosed to have it. When used to monitor the status of a cancer, aquantitative immunoassay procedure must be used. If such monitoringassays are carried out periodically and the results compared, adetermination may be made regarding whether the patient's tumor burdenhas increased or decreased. Common assay techniques that may be usedinclude direct and indirect assays. If the sample includes cancer cells,the labeled antibody will bind to those cells. After washing the tissueor cells to remove unbound labeled antibody, the tissue sample is readfor the presence of labeled immune complexes. In indirect assays thetissue or cell sample is incubated with unlabeled monoclonal antibody.The sample is then treated with a labeled antibody against themonoclonal antibody (e.g., a labeled antimurine antibody), washed, andread for the presence of ternary complexes.

For diagnostic use the antibodies will typically be distributed in kitform. These kits will typically comprise: the antibody in labeled orunlabeled form in suitable containers, reagents for the incubations foran indirect assay, and substrates or derivatizing agents depending onthe nature of the label. HER2 p185 controls and instructions may also beincluded.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

Experimental

Amplified Expression of p185^(HER2) and Tyrosine Kinase Activity

A series of NIH 3T3 cell lines expressing various levels of p185 wereconstructed as disclosed in Hudziak et al., Proc. Natl. Acad. Sci. (USA)84, 7159 (1987), hereby incorporated by reference. The parental cellline had a nontransformed, TNF-α-sensitive phenotype. The control cellline (NIH 3T3 neo/dhfr) was prepared by transfection with pCVN, anexpression plasmid encoding neomycin resistance as a selectable marker,and dihydrofolate reductase (which encodes methotrexate resistance andwhich permits amplification of associated DNA sequences). pCVN-HER2(which encodes, in addition, the entire 1255 amino acid p185receptor-like tyrosine kinase under the transcriptional control of theRSV-LTR) was introduced into NIH 3T3 cells in a parallel transfection.Transfectants were selected by resistance to the aminoglycosideantibiotic G418. The pCVN-HER2 primary transfectants (HER2-3) do nothave a transformed morphology and fail to grow in soft agar. Stepwiseamplification of HER2 expression by selection in 200 nM (HER2-3₂₀₀), 400nM (HER2-3₄₀₀), and 800 nM (HER2-3₈₀₀) methotrexate, however, results intransformation as judged by morphological criteria, the ability to growin soft agar, and the ability to form tumors in nude mice.

The amplification of expression of p185 was documented byimmunoprecipitation from cells that were metabolically labeled with³⁵S-methionine. The tyrosine kinase activity associated with p185 inthese cell lines was measured by autophosphorylation in vitro. For anautoradiograph of ³⁵S-methionine labeled p185, 200 μCi of ³⁵S-methionine(Amersham; 1132 Ci/mmol) was added to 1.5 ml of methionine-free labelingmedium, containing 2% dialyzed fetal bovine serum. 1.0×10⁶ cells of eachtype were counted by Coulter counter, plated in 60 mm culture dishes(Falcon), and allowed to adhere for 12 h. Following an 8 h labelingperiod the cells were lysed and the HER2-encoded p185 was analyzed. Foran autoradiograph of self-phosphorylated HER2-receptor tyrosine kinase,the p185 was immunoprecipitated and the pellet was resuspended in 50 μlof tyrosine kinase reaction buffer. The samples were incubated at 4° C.for 20 min. The self-phosphorylated p185 from the various cell lines wasthen visualized by autoradiography following gel electrophoresis. Themolecular weight markers used were myosin (200 kD) and β-galactosidase(116 kD). The results showed that expression of p185 and its associatedtyrosine kinase increased in parallel during amplification. Quantitativedensitometry of the in vitro autophosphorylation reactions showed thatthe tyrosine kinase activity increased at least 5 to 6-fold betweenHER2-3 and HER2-3₂₀₀ and between HER2-3₂₀₀ and HER2-3₄₀₀, while only asmall difference was observed between HER2-3₄₀₀ and HER2-3₈₀₀ (see theTyrosine Kinase column of Table 1 below).

Relative amounts of tyrosine kinase present in each of the cell types ofTable 1 were determined by taking ratios of the areas under the curvesobtained by scanning autoradiograms (using an LKB2202 laserdensitometer). The autoradiograms had been exposed for various times toallow for linearity in the determinations, and then normalized bycomparison to the HER2 primary transfectant (HER2-3).

Resistance to TNF-α

The cell lines described above were then tested for sensitivity to TNF-αand macrophage-induced cytotoxicity.

In FIG. 1a, TNF-α resistance of the control cells and theHER2-transfected NIH 3T3 cells is shown. Cells were seeded into 96-wellmicrotiter plates at a density of 5,000 cells/well in DMEM supplementedwith 10% calf serum, 2mM L-glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin. The cells were allowed to adhere for 4 hrs before theaddition of a range of concentrations of TNF-α. Specific activity of theTNF-α (recombinant human TNF-α) was 5×10⁷ U/mg as determined in an L-Mcell cytotoxicity assay in the presence of actinomycin D. Afterincubation at 37° C. for 72 hr, the monolayers were washed with PBS andstained with crystal violet dye for determination of relative cellviability. These measurements were repeated six times. Results from arepresentative experiment are shown in FIG. 1a.

In FIG. 1b, macrophage-mediated cytotoxicity assays are shown. TNF-αresistant cells (neo/dhfr HTR) were derived by subculturing a clone ofNIH 3T3 neo/dhfr in media containing 10,000 U/ml TNF-α. For macrophagecytotoxicity assays, NIH 3T3 neo/dhfr, HER2-3₈₀₀ and neo/dhfr HTR cellswere seeded into 96-well microtiter plates as in 1 a above. Humanmacrophages were obtained as adherent cells from peripheral blood ofhealthy donors. Adherent cells were scraped and resuspended in media,activated for 4 hr. with 10 μg/ml E. coli-derived lipopolysaccharide(LPS; Sigma) and 100 U/ml of recombinant human interferon-gamma(rHuIFN-γ, Genentech, Inc.). The cell suspension was then centrifugedfor 10 minutes at 1200 rpm and the resulting pellet was washed withmedia to remove the LPS and rHuIFN-γ. The macrophages were resuspendedin media, counted, and then added to the target cells to obtain thedesired effector to target ratios. After a 72 hr incubation at 37° C.,the monolayers were washed with media and ⁵Cr was added to each well fordetermination of viability by ⁵¹Cr uptake.

TABLE 1 Correlation between HER2-associated tyrosine kinase levels andresistance to TNF-α Percent Tyrosine Cell Type Viability Kinase  1. NIH3T3 neo/dhfr  3.6 ± 0.6 *  2. NIH 3T3 neo/dhfr₄₀₀  8.3 ± 1.0 *  3.HER2-3  2.0 ± 0.4 1.0  4. HER2-3₂₀₀ 27.5 ± 2.7 6.73  5. HER2-3₄₀₀ 48.4 ±1.4 32.48  6. HER2-3₈₀₀ 58.7 ± 1.3 39.61  7. BT-20  1.6 ± 0.3 <0.1  8.MCF7  2.5 ± 0.3 0.26  9. MDA-MB-361 26.8 ± 6.6 10.65 10. MDA-MB-175-VII31.2 ± 4.4 0.9 11. SK-BR-3 56.4 ± 5.5 31.0 12. MDA-MB-231 64.2 ± 9.3<0.1 * not measured

Percent viability is given at 1.0×10⁴ cytotoxicity units per ml ofTNF-α. The breast tumor cell lines were obtained from the ATCC andmaintained in DMEM supplemented with 10% fetal bovine serum, 2 mMglutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

As shown in FIG. 1a and Table 1, stepwise amplification of HER2 receptorexpression resulted in a parallel induction of resistance to TNF-α. Theprimary transfectants (HER2-3), which do not have a transformedphenotype, demonstrated little increased resistance. However, thetransformed lines HER2-3₂₀₀, HER2-3₄₀₀ and HER2-3₈₀₀ do show a stepwiseloss in sensitivity to TNF-α-mediated cytotoxicity as compared to NIH3T3 neo/dhfr (FIG. 1a and Table 1), although the MDA-MB-175-VII cellshad elevated p185 expression compared to the TNF-α sensitive BT20 andMCF7 cell lines. In correlation with the levels of p185 expression(Table 1), the difference in sensitivity of HER2-3₂₀₀ and HER2-3₄₀₀(27.5% vs. 48.4% viability at 1×10⁴ U/ml TNF-α) is greater than thedifference between HER2-3₄₀₀ and HER2-3₈₀₀ (48.4% vs. 58.7% viability,see FIG. 1a and Table 1). A similar result was obtained when NIH 3T3neo/dhfr and HER2-3₈₀₀ were compared for sensitivity to activatedmacrophages (FIG. 1b). These data suggest that amplification of theexpression of HER2 induces resistance to TNF-α, and also show that thiscorrelates with resistance to an important component of the early hostdefense mechanism, the activated macrophage. Amplification of thecontrol plasmid (pCVN) in the cell line NIH 3T3 neo/dhfr₄₀₀ did notinduce increased resistance to TNF-α (Table 1). This demonstrates thatneither gene transfection or gene amplification, per se, has any effecton the sensitivity of cells to TNF-α.

The observation that NIH 3T3 cell lines expressing high levels of p185were resistant to cytotoxicity induced by TNF-α or macrophages suggestedthat this may be one mechanism leading to tumor development. To testthis possibility six breast tumor cell lines were screened foramplification of HER2 and sensitivity to TNF-α-mediated cytotoxicity.The results (Table 1) demonstrated that sensitivity to growth inhibitionby TNF-α is inversely correlated with the expression of HER2-associatedtyrosine kinase measured in vitro autophosphorylation assay for BT-20,MCF7, MDA-MB-361 and SK-BR-3. Two of the TNF-α-resistant breast tumorcell lines (MDA-MB-175-VII and MDA-MB-231), however, had no demonstrableamplified expression of HER2 as compared to the HER2-3 control (Table1), although the MDA-MB-175-VII cells had elevated p185 expressioncompared to the TNF-α sensitive BT20 and MCF7 cell lines. These resultsare consistent with previous reports of the frequency of HER2amplification in primary breast tumors and tumor-derived cell lines, andsuggest the existence of other cellular mechanisms which may lead toTNF-α resistance.

Experiments also showed that overexpression of the EGF receptor, andcellular transformation by the src oncogene, correlates with resistanceto TNF-α.

TNF-α Receptor Binding

In order to investigate whether the TNF-α receptor was altered inHER2-3₈₀₀, as opposed to NIH 3T3 neo/dhfr, the binding of ¹²⁵I-labeledTNF-α was compared between these cell lines. FIG. 2 shows a TNF-αreceptor binding analysis. Displacement curves show binding of¹²⁵I-TNF-α to NIH 3T3 neo/dhfr and HER2-₃₈₀. Competition binding assayswere performed. Briefly, a suspension of 2.0×10⁶ cells were incubated ina final volume of 0.5 ml of RPMI-1640 medium containing 10% fetal bovineserum. Binding of ¹²⁵I-TNF-α (0.2×10⁶ cpm) to the cells was determinedin the presence or absence of varying concentrations of unlabeled TNF-αat 4° C. under saturation equilibrium conditions. Each data pointrepresents the mean of triplicate determinations. After incubationovernight, cells were washed twice with incubation buffer and cell boundradioactivity was determined. Non-specific binding was <10% of totalbinding.

The results showed a 2-3 fold increase in total specific binding forHER2-3₈₀₀ as compared to NIH 3T3 neo/dhfr (FIG. 2). In addition, thedisplacement curve for binding on HER2-3₈₀₀ is also shifted toward loweraffinity binding as compared to NIH 3T3 neo/dhfr (FIG. 2).

Production of Anti-HER2 Monoclonal Antibodies

Five female Balb/c mice were immunized with HER2 amplified NIH 3T3transformed cells over a period of 22 weeks. The first four injectionseach had approximately 10⁷ cells/mouse. They were administeredintraperitoneally in half a milliliter of PBS on weeks 0, 2, 5, 7.Injections five and six were with a wheat germ agglutinin partiallypurified membrane preparation which had a whole protein concentration ofabout 700 μg/ml. A 100 μl/injection was administered to each mouseintraperitoneally on weeks 9 and 13. The last injection was also withthe purified material but was administered three days prior to the dateof fusion intravenously.

Bleeds from the mice were tested at various times in aradioimmunoprecipitation using whole cell lysates. The three mice withthe highest antibody titers were sacrificed and spleens were fused withthe mouse myeloma cell line X63-Ag8.653 using the general procedure ofMishell & Shiigi, Selected Methods in Cellular Immunology, W. H. Freeman& Co., San Francisco, p. 357-363 (1980) with the following exceptions.Cells were plated at a density of approximately 2×10⁵ cells/well intoten 96 well microtiter plates. Hybrids were selected usinghypoxanthine-azoserine rather than hypoxanthine-aminoptern-thymidine(HAT).

Hybridoma supernatants were tested for presence of antibodies specificfor HER2 receptor by ELISA and radioimmunoprecipitation.

For the ELISA, 3.5 μg/ml of the HER2 receptor (purified on the wheatgerm agglutinin column) in PBS was adsorbed to immulon II microtiterplates overnight at 4° C. or for 2 hours at room temperature. Plateswere then washed with phosphate buffered saline with 0.05% Tween 20(PBS-TW20) to remove unbound antigen. Remaining binding sites were thenblocked with 200 μl per well of 1% bovine serum albumin (BSA) inPBS-TW20 and incubated 1 hour at room temperature. Plates were washed asabove and 100 μl of hybridoma supernatant was added to each well andincubated for 1 hour at room temperature. Plates were washed again and100 μl per well of an appropriate dilution of goat anti-mouseimmunoglobulin coupled to horseradish peroxidase was added. The plateswere incubated again for 1 hour at room temperature and then washed asabove. O-phenylene diamine was added as substrate, incubated for 15-20minutes at room temperature and then the reaction was stopped with 2.5 MH₂SO₄. The absorbance of each well was then read at 492 nm.

For the radioimmunoprecipitation, first the wheat germ purified HER2rceptor preparation was autophosphorylated in the following manner: akinase solution with the following final concentrations was made: 0.18mCi/ml γP³²-ATP (Amersham), 0.4 mM MgCl₂, 0.2 mM MnCl₂, 10 μM ATP, 35μg/ml total protein concentration of partially purified HER2 all dilutedin 20 mM Hepes, 0.1% triton 10% glycerol buffer (HTG). This reaction wasincubated for 30 minutes at room temperature. 50 μl hybridomasupernatant was then added to 50 μl of the kinase reaction and incubated1 hour at room temperature. 50 μl of goat anti-mouse IgC precoatedprotein-A sepharose CL4B, at a sepharose concentration of 80 mg/ml, wasadded to each sample and incubated 1 hour at room temperature. Theresulting immunocomplexes were then washed by centrifugation twice withHTG buffer and finally with 0.2% deoxycholate 0.2% Tween 20 in PBS, in amicrofuge and aspirated between washes. Reducing sample buffer was addedto each sample and samples were heated at 95° C. for 2-5 minutes,insoluble material was removed by centrifugation and the reducedimmunocomplex was loaded onto a 7.5% polyacrylamide gel containing SDS.The gel was run at 30 amp constant current and an autoradiograph wasobtained from the finished gel.

Approximately 5% of the total well supernatants reacted with the HER2receptor in the ELISA and/or radioimmunoprecipitation. From this initial5% (about 100), some hybrids produced low affinity antibodies and otherssuffered from instability and stopped secreting antibodies leaving atotal of 10 high affinity stable HER2 specific antibody producing celllines. These were expanded and cloned by limiting dilution (Oi, V.T. andHerzenberg, L.A., “Immunoglobulin Producing Hybrid Cell Lines” inSelected Methods in Cellular Immunology, p. 351-372 Mishell, B. B. andShiigi, S. M. (eds.), W. H. Freeman and Co. (1980)). Large quantities ofspecific monoclonal antibodies were produced by injection of clonedhybridoma cells in pristaned primed mice to produce ascitic tumors.Ascites were then collected and purified over a protein-A sepharosecolumn.

Screening of Antibodies

The 10 high affinity monoclonal antibodies were then screened in anumber of assays for anti-transformation or anti-tumor cell activity.Monoclonal antibodies were selected on the basis of growth inhibitingactivity against the human tumor line SK BR3 which is derived from abreast tumor and contains an amplified HER2 gene and overexpresses theHER2 p185 tyrosine kinase. The initial screen used conditioned medium(medium in which the cells were grown for several days containing anysecreted products of the cells including antibodies produced by thecells) from the hybridoma cell lines.

SK BR3 cells were plated at 20,000 cells/35 mm dish. Either conditionedmedium from the hybridoma parent line (producing everything butanti-HER2 monoclonals) as a control, or the anti-HER2 monoclonals wereadded. After 6 days, the total number of SK BR3 cells were counted usingan electronic Coulter cell counter. Cells were grown in a 1:1 mixture ofF-12 and DMEM supplemented with 10% fetal bovine serum, glutamine, andpenicillin-streptomycin. The volume per plate was 2 mls/35 mm dish. 0.2mls of myeloma conditioned medium was added per 35 mm dish. Each controlor anti-HER2 MAb was assayed in duplicate and the two counts averaged.

The result of the survey is shown in FIG. 3. Monoclonal antibody 4D5(ATCC CRL 10463, deposited with American Type Culture Collection,10801University Blvd., Manassas, Va. 20110-2209 under the Budapest Treaty onMay 24, 1990) markedly inhibited the growth of the breast tumor line SKBR3. Other anti-HER2 antibodies inhibited growth to a significant butlesser extent (egs., MAbs 3E8 and 3H4). Still other anti-HER2,antibodies (not shown) did not inhibit growth.

A repeat experiment using purified antibody rather than hybridomaconditioned medium confirmed the results of FIG. 3. FIG. 4 is a doseresponse curve comparing the effect of an irrelevant monoclonal antibody(anti-HBV) and monoclonal antibody 4D5 (anti-HER2) on the growth of theSK BR3 cell line in 10% fetal bovine serum.

Down Regulation of the HER2 Receptor

The SK BR3 cells were pulse labeled with 100 uci ³⁵S-methionine for 12hours in methionine-free medium. Then either an irrelevant(anti-Hepatitis B surface antigen) or anti-HER2 MAb (4D5) was added tothe cells at 5 μg/ml. After 11 hours, the cells were lysed and HER2 p185immunoprecipitates and the proteins were analyzed by a 7.5% acrylamidegel followed by autoradiography. The SDS-PAGE gel of the ³⁵S-methioninelabeled HER2 p185 from SK BR3 cells demonstrated that the HER2 levelsare downregulated by MAb 4D5.

Treatment of Breast Tumor Cells with Monoclonal Antibodies and TNF-α

SK-BR-3 breast tumor cells were seeded at a density of 4×10⁴ cells perwell in 96-well microtiter plates and allowed to adhere for 2 hours. Thecells were then treated with different concentrations of anti-HER2monoclonal antibody (MAb) 4D5 or irrelevant isotype matched(anti-rHuIFN-γ MAb) at 0.05, 0.5 or 5.0 μg/ml for 4 hours prior to theaddition of 100, 1,000 or 10,000 U/ml rHuTNF-α. After a 72 hourincubation, the cell monolayers were stained with crystal violet dye fordetermination of relative percent viability (RPV) compared to control(untreated) cells. Each treatment group consisted of 6 replicates. Theresults are shown in FIGS. 5 and 6. These Figures show that incubationof cells overexpressing HER2 receptor with antibodies directed to theextracellular domain of the receptor induce sensitivity to the cytotoxiceffects of TNF-α. Equivalent treatment of breast tumor cells MDA-MB-175VII gave similar results (see FIG. 7). Treatment of human fetal lungfibroblasts (WI-38) with MAb resulted in no growth inhibition orinduction of sensitivity to TNF-α as expected.

Treatment of NIH 3T3 Cells Overexpressing HER2 p185 with MonoclonalAntibodies and TNF-α

NIH 3T3 HER2-3₄₀₀ cells were treated with different concentrations ofanti-HER2 MAbs as in the above described treatment of SK-BR3 cells. Theresults for MAb 4D5 are shown in FIG. 8. The results indicate that cellsother than of breast tumor cell lines which overexpress the HER2receptor are growth inhibited by antibodies to the HER2 receptor, andsenstivity to TNF-α is induced in the presence of these antibodies.

In Vivo Treatment of NIH 3T3 Cells Overexpressing HER2 with Anti-HER2IgG2A Monoclonal Antibodies

NIH 3T3 cells transfected with either a HER2 expression plasmid (NIH3T3₄₀₀) or the neo-DHFR vector were injected into nu/nu (athymic) micesubcutaneously at a dose of 10⁶ cells in 0.1 ml of phosphate-bufferedsaline. On days 0, 1, 5 and every 4 days thereafter, 100 μg (0.1 ml inPBS) of either an irrelevant or anti-HER2 monoclonal antibody of theIgG2A subclass was injected intraperitoneally. Tumor occurence and sizewere monitored for the 1 month period of treatment.

TABLE 2 Tumor Size of Survivors: Length × Width # Tumors/ Average in mm²Group # Cell Line Treatment # Animals at 31 Days 1 HER2 (3T3₄₀₀)Irrelevant 6/6 401 MAb (anti- Hepatitis B Virus) 2 HER2 (3T3₄₀₀) 2H11anti- 2/6 139 HER2 3 HER2 (3T3₄₀₀) 3E8 anti- 0/6 0 HER2 4 neo/DHFR None0/6 0

Table 2 shows that the 2H11 MAb has some anti-tumor activity (MAb 2H11has very slight growth inhibiting properties when screened against tumorline SK BR3) and the 3E8 MAb gives 100% tumor growth inhibition duringthe course of the experiment.

While the invention has been described in what is considered to be itspreferred embodiments, it is not to be limited to the disclosedembodiments, but on the contrary, is intended to cover variousmodifications and equivalents included within the spirit and scope ofthe appended claims, which scope is to be accorded the broadestinterpretation so as to encompass all such modifications andequivalents.

What is claimed is:
 1. A method of inhibiting the growth of tumor cellsthat overexpress HER2 receptor comprising administering to a patient anantibody comprising an antigen binding region which specifically bindsto an extracellular domain of the HER2 receptor in an amount effectiveto inhibit growth of the tumor cells in the patient, wherein theantibody is not conjugated to a cytotoxic moiety.
 2. A method oftreating cancer that overexpresses HER2 receptor comprisingadministering to a patient an antibody comprising an antigen bindingregion which specifically binds to an extracellular domain of the HER2receptor in an amount effective to eliminate or reduce the patient'stumor burden, wherein the antibody is not conjugated to a cytotoxicmoiety.
 3. The method of claim 2 wherein the patient has breast cancer.4. A method of treating renal cancer that overexpresses HER2 receptorcomprising administering to a patient an antibody comprising an antigenbinding region which specifically binds to an extracellular domain ofthe HER2 receptor in an amount effective to eliminate or reduce thepatient's tumor burden.
 5. A method of treating gastric cancer thatoverexpresses HER2 receptor comprising administering to a patient anantibody comprising an antigen binding region which specifically bindsto an extracellular domain of the HER2 receptor in an amount effectiveto eliminate or reduce the patient's tumor burden.
 6. A method oftreating salivary gland cancer that overexpresses HER2 receptorcomprising administering to a patient an antibody comprising an antigenbinding region which specifically binds to an extracellular domain ofthe HER2 receptor in an amount effective to eliminate or reduce thepatient's tumor burden.
 7. A method of treating cancer comprisingidentifying a patient with cancer characterized by amplification of theHER2 gene and/or overexpression of the HER2 receptor and administeringto the patient thus identified an antibody comprising an antigen bindingregion which specifically binds to an extracellular domain of the HER2receptor in an amount effective to inhibit growth of the cancer of thepatient.
 8. A method of treating cancer that overexpresses HER2 receptorcomprising administering to a patient an effective amount of an antibodycomprising an antigen binding region which specifically binds to anextracellular domain of the HER2 receptor and an effective amount of acytokine, wherein administration of the antibody and the cytokine to thepatient inhibits growth of the cancer in the patient.
 9. The method ofclaim 8 wherein the cytokine is tumor necrosis factor-α (TNF-α).
 10. Themethod of claim 8 wherein the cytokine is tumor necrosis factorβ(TNF-β).
 11. The method of claim 8 wherein the cytokine is interleukin-1(IL-1).
 12. The method of claim 8 wherein the cytokine is interleukin-2(IL-2).
 13. The method of claim 8 wherein the cytokine is interferon-γ(IFN-γ).
 14. The method of claim 8 wherein the antibody and cytokine areadministered to the patient together.
 15. The method of claim 8 whereinthe antibody and cytokine are administered to the patient separately.16. The method of claim 8 wherein the antibody binds to the HER2receptor epitope to which antibody 4D5 (ATCC CRL 10463) binds.
 17. Themethod of claim 8 wherein the antibody has the identifying biologicalcharacteristics of antibody 4D5 (ATCC CRL 10463).
 18. The method ofclaim 8 wherein the antibody interacts with antibody dependent cytotoxiccells.